ppRB110 - nuclear phosphoprotein - the retinoblastoma susceptibility gene product

ABSTRACT

This invention relates in general to a phosphoprotein product of the retinoblastoma susceptibility gene. In particular, this invention relates to a phosphoprotein ppRB 110  primarily located in the cell nucleus which has a DNA binding activity. The invention also relates to the amino acid sequence of the phosphoprotein and to the specific purified anti-retinoblastoma phosphoprotein antibody. The invention further relates to a method of diagnosing retinoblastoma and other retinoblastoma gene involved cancers, treating such kind of cancers and regulating the oncogenicity of other genes.

This invention was made with Government support under grant No.: EY05758 with the National Institute of Health and the University ofCalifornia. The Government has certain rights in this invention.

This application is a continuation of U.S. Ser. No. 08/079,207, filedJun. 17, 1993 abandoned, which is a continuation of U.S. Ser. No.07/914,039, filed Jul. 14, 1992, now abandoned, which is a continuationof U.S. Ser. No. 07/550,877, filed Jul. 11, 1990, now abandoned, whichis a divisional of U.S. Ser. No. 07/098,612, filed Sep. 17, 1987, issuedas U.S. Pat. No. 4,942,123.

FIELD OF THE INVENTION

This invention relates in general to a phosphoprotein product of theretinoblastoma susceptibility gene. In particular, this inventionrelates to a phosphoprotein ppRB¹¹⁰ primarily located in the cellnucleus which has a DNA binding activity. The invention also relates tothe amino acid sequence of the phosphoprotein and to the specificpurified anti-retinoblastoma phosphoprotein antibody. The inventionfurther relates to a method of diagnosing retinoblastoma and otherretinoblastoma gene involved cancers, treating such kind of cancers andregulating the oncogenicity of other genes.

Background

Retinoblastoma is a malignant tumor of the sensory layer of the retina.The neoplastic tumor is composed of primitive retinal cells, occurringoften bilaterally, usually before the third year of life. It exhibits afamilial tendency. Retinoblastomas are characterized by small roundcells with deeply staining nuclei, and elongated cells forming rosettes.They usually cause death by local invasion, especially along the opticnerves.

The retinoblastoma may be hereditary but also acquired. It is the mostcommon intraocular tumor and represents one of the prototypes ofinheritable cancers. The hereditary form is characterized by early ageof onset and multiple tumor foci. Acquired form occurs later in lifewith a single unilateral tumor (Proc. Natl. Acad. Sci., 68:820-823[1971]; Hum. Genet., 52:1-54 [1979]; Science, 223:1028-1033 [1984]).

The molecular mechanism of the formation of this tumor is unknown.Absence or inactivation of the retinoblastoma (RB) gene is believed tobe the primary cause of this cancer (Science, 213:1501-1503 [1981];Science, 223:1028-1033 [1984]; Proc. Natl. Acad. Sci., 68:820-823[1971]; Nature, 305:779-784 [1980]).

Susceptibility to hereditary retinoblastoma is transmissible tooffsprings as an autosomal dominant trait with 90% penetrance, and thetumor is, therefore, a prototypic model for the study of geneticdetermination in cancer (Am. J. Hum. Genet., 30:406-410 [1978]; Cancer,35:1022-1026 [1975]).

There are at least two hypotheses related to the oncogenesis ofretinoblastoma. The first hypothesis suggests that the tumor is causedby two mutational events (Proc. Natl. Acad. Sci., 68:820-823 [1971];Cancer, 35:1022-1026 [1975]). The other hypothesis proposes thatautosomal dominant hereditary tumors, such as retinoblastoma, representthe inheritance of a defective regulatory or suppressor gene whichnormally regulates a group of transforming genes, most probablyprotooncogenes. Such genes, when active, would release the cell from itsnormal constraints on growth and therefore, if and when somatic mutationinactivates the normal suppressor gene, such as for example the RB gene,tumors can develop (Proc. Natl. Acad. Sci., 70:3324-3328 [1973]).

Based on these hypotheses, hereditary retinoblastoma might arise from aprecursor retinoblast cell, carrying one inherited defective allelewhich suffers an additional somatic mutation, while nonhereditary caseswould require two somatic mutations in the same cell. Recentcircumstantial evidence supports the existence of such cancer suppressorgenes in retinoblastoma (Nature, 305:779-781 [1983]; Proc. Natl. Acad.Sci., 83:7391-7394 [1986]; Science, 235:1394-1399 [1987]) as well asnephroblastoma also known as Wilm's tumor (Nature, 309:172 -174 [1984];Nature, 309:176-178 [1984]; neuroblastoma (Cancer Res., 41:4678-4682[1981]), osteosarcoma (Proc. Natl. Acad. Sci., 82:6216-6220);retinoblastoma (Hum. Genet., 52:1-54 [1979]; Ann. Hum. Genet.,27:171-174 [1963]; Am. J. Dis. Child, 132:161-163 [1978]; Science,208:1042-1044 [1980]; Science, 213:1501-1503 [1981]). Linkage wasestablished to the gene for the polymorphic marker enzyme esterase D,which also maps to 13q14 (Science, 219:971-973 [1983]).

Additional evidence supporting this genetic assignment came from thepedigree of a family showing balanced chromosomal translocations inunaffected carrier parents and in some unaffected siblings but with anunbalanced chromosome 13 deletion in affected individuals (Science,213:1501-1503 [1981]). This observations also indicated that theretinoblastoma susceptibility locus is able to function in the "trans"configuration.

The RB locus was further implicated in non-hereditary retinoblastoma byobserving frequent abnormalities of chromosome 13 in tumor karyotypesand reduced esterase D activity in tumors (Cancer Genet. Cytogenet.,10:311-333 [1983]; Cancer Genet. Cytogenet., 6:213-221 [1982]). It hasbeen proposed that inactivation of both alleles of the RB gene locatedin region 13q14 resulted in retinoblastoma. Such proposal was based inpart on a case of hereditary retinoblastoma in which both RB alleleswere inferred to be absent (Science, 219:973-975 [1983]). However, theassumption upon which this proposal was based, namely that the absenceof esterase D activity in this case implied loss of both esterase D andRB genes, has been disproved (Hum. Genet., 76:33-40 [1987]).Nonetheless, the other findings show that chromosome 13 markers whichwere heterozygous in somatic cells often became homozygous or hemizygousin retinoblastoma tumors, and that there are homozygous deletions in the13q14 region in 3/37 retinoblastoma cell lines (Nature, 305:779-784[1983]; Proc. Natl. Acad. Sci., 83:7391-7394 [1986]). These experimentsprovide evidence that the proposed RB gene indeed functions in arecessive manner at the cellular level (Science, 235:305-311 [1987];Cancer Res., 46:1573-1580 [1986]) in distinction to the "dominant"activity of classical oncogenes (Science, 228:669-676 [1985]; Nature,315:190-195 [1985]) as measured, for example, by transfection assays.

Thus, while an extensive research effort is devoted to inherited andacquired retinoblastoma and to its causes, reliable methods are notreadily available for diagnosing the embryo, fetus, or newborn forinherited retinoblastoma or the child or adult for predisposition todevelop acquired retinoblastoma or secondary tumors that often accompanyretinoblastoma. This is true mainly because the protein intermediatingthe function of the RB gene has not been known, isolated or identified.

Therefore, it would be advantageous to acquire more knowledge about themolecular and chemical properties of the retinoblastoma gene throughcloning and isolation of the gene, identification of the gene sequenceand by gene mapping. Isolation of the RB gene's protein product and theidentification of its amino acid sequence would also be advantageous aswell as preparation of specific anti-RB protein antibody, particularlybecause the RB gene function is intermediated by the specific RB proteinwhich, in turn, can only be recognized in the tissue by the specificanti-RB protein antibody.

Both forms of retinoblastoma can now be treated and most patients can befollowed through adult life. However, patients with hereditaryretinoblastoma have an extraordinarily high risk for developingsecondary nonocular malignancies (Ophthalmology, 91:1351-1355 [1984];New Engl. J. Med., 285:307-311 [1971]; Cancer, 34:2077-2079 [1974]; upto 90% incidence within 30 years of initial diagnosis (Ophthalmology,91: 131-136 [1984]). The most frequently occuring secondary cancer isosteosarcoma, which is otherwise uncommon. In contrast, curednonhereditary retinoblastoma patients show the same cancer rates as thegeneral population. This finding is of considerable interest, since itimplies that the RB gene may have a critical role in regulating othertumors as well.

Without complete isolation and identification of the RB gene and itsprotein product, an early diagnosis of the osteosarcoma or other RBsecondary tumors, or the predisposition thereto, is unavailable.

Therefore, it would be advantageous to have available a means todiagnose the primary retinoblastoma or the susceptibility thereto, andto provide timely forewarning against the secondary molecularmalignancies. The best way to achieve the above goals, is by theidentification of the amino acid sequence of the RB gene productprotein, which protein, per se, is the intermediator of the RB generegulatory function not only for retinoblastoma but also for itssecondary tumorigenic regulating activity.

It is of obvious importance to understand the molecular nature of the RBgene, and the mechanism of its regulatory function through the proteinproduct produced by retinoblastoma gene.

Recently, human retinoblastoma gene was successfully cloned, identifiedand sequenced (Science 235:1394-1399 [1987]). The retinoblastoma genewas located in the chromosome 13 region 13q14:11 in the close proximityof the esterase D gene, also recently identified, cloned and sequenced(Proc. Natl. Acad. Sci., 83:6790-6794 [1986]; Proc. Natl. Acad. Sci.,83:6337-6341 [1986]). By chromosomal walking from esterase D genes, theretinoblastoma (RB) gene was identified on the basis of chromosomallocation, homozygous deletion and tumorspecific alterations inexpression. RB gene was shown to have 4723 nucleotides and encodes amessenger RNA (mRNA) of 4.8 kilobases (kb).

Transcription of RB DNA to RB mRNA was found to be abnormal inretinoblastoma patients. Transcription was either not detected at all,suggesting the absence or complete inactivation of the RB gene, ortranscribed mRNA had shown decreased molecular size of about 4.0 kb,suggesting defective RB gene.

Sequence of RB complementary DNA (cDNA) clones yielded a single longopen reading frame suggesting that it could encode a hypotheticalprotein of 816 amino acid. A computer-assisted search of a proteinsequence data base revealed no closely related proteins suggesting aunique amino acid sequence of the predicted protein (Science,235:1394-1399 [1987]). The predicted protein will seem to have severalproline rich regions, similar to those previously observed in othernuclear oncogenes proteins such as proteins "myc" and "myb" (RNA TumorViruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.[1985]).

The hypothetical amino acid composition of the RB gene product proteinseems to contain several distinguishable regions which were shown to besimilar to the other oncogenic proteins. This finding suggests that theRB gene protein product could have similar regulatory functions as theseother oncogene proteins.

However, without chemical and molecular characterization on the RB geneprotein product and without specific anti-RB protein antibody, thefurther elucidation of the RB protein in regulation of tumorigenesis isimpossible.

Therefore, it would be advantageous to identify the amino acid sequenceof the RB gene, to determine its subcellular localization and todetermine whether it has DNA binding activity.

It would also be advantageous to obtain the exact amino acid sequence ofthe RB gene product, to prepare or isolate the portion or the whole RBprotein in purified form, and to obtain a specific anti-retinoblastomaprotein antibody which would specifically recognize the retinoblastomaprotein in the tissue. Such antibody would then be employed as adiagnostic tool to recognize the presence or the absence of the RB geneprotein product. Thus, the anti-RB protein antibody would diagnose thenormal or abnormal protein of the RB gene in several different kinds ofhuman cancer.

It is, therefore, the object of this invention to provide a completeamino acid sequence of the RB gene protein product.

It is another object of this invention to biochemically characterize theRB gene protein product and to determine its molecular weight.

It is still another object of this invention to determine thesubcellular localization of the RB gene protein product.

It is yet another object of this invention to provide a metabolicallylabeled radioactive RB gene protein product.

It is a further object of this invention to provide a specificanti-retinoblastoma protein polyclonal antibody.

It is still a further object of this invention to provide a diagnosticmethod for hereditary predisposition to retinoblastoma in fetus, embryoand newborn, or for the susceptibility in the later age to acquire asecondary cancer associated with the retinoblastoma. Example ofretinoblastoma gene involved cancers are osteosarcoma, fibrosarcoma,glioblastoma and breast cancer.

It is still a further object of this invention to provide a method fortreatment of cancerous growth through the regulation of growth promotinggenes and by genetic manipulation.

DETAILED DISCLOSURE OF THE INVENTION

All references cited in this application are hereby incorporated byreference and made part of this application.

Experimental evidence indicates that complete inactivation of the RBgene is required for tumor formation, and hence indicates a new mode offunction for the RB gene as a suppressor of the cancer phenotype.

Since the gene action is generally intermediated by its protein product,it appears that THE RB gene protein product would have a gene-regulatoryactivity.

Therefore, obtaining the complete Amino acid sequence of the RB geneprotein product, specific antiretinoblastoma protein antibody, itsbiochemical characterization, subcellular localization and its DNAbinding activity, would be of utmost importance for further elucidationof the RB gene regulatory and oncogenic activity.

The complete amino acid sequence of the retinoblastoma gene protein isthe subject of previously noted article in Science, 235:1394-1399[1987]. The specific polyclonal anti-retinoblastoma protein antibody wasprepared. The RB protein was localized by subcellular fractionation andits evolutionary conservation was shown. DNA binding activity of the RBgene protein was proven.

Amino acid sequence of any protein is determined by the genetic code ofthe particular gene responsible for that particular protein. Therefore,in order to isolate the protein, to determine its exact amino acidsequence and to determine its physiological function in the body, it isnecessary to isolate and localize the responsible gene, to clone it andto sequence the cDNA which are useful in identification of the gene'sspecific protein product.

Using the method of chromosomal walking from other chromosome 13markers, retinoblastoma gene and encoding of the amino acid sequence wasidentified at chromosome, 13 q14:11 region. By using esterase D cDNAclones and by screening the genomic and cDNA libraries, several cloneswere obtained. From these clones, two cDNA overlapping clones RB-1 andRB-2 of 1.6 kb and 0.9 kb, respectively, were identified in human cDNAlibraries. Later on, another clone RB-5 was also identified.

In the process of developing this invention, first, the RB-1 clone washybridized with a 4.8 kb mRNA transcript in human fetal retina andplacenta libraries. In retinoblastoma samples, RB-1 clone eitherdetected an abnormal mRNA transcript or the mRNA transcripts were notobserved at all. Subsequently identified RB-5 clone, with a 3.5 kbinsert, gave identical results as RB-1 in mRNA hybridization.Restriction enzyme analysis suggested that RB-5 and RB-1 clonesoverlapped in a 0.4 kb region and both together defined a DNA segment ofabout 4.6 kb, a size close to that of the normal RB mRNA transcript.

Nucleotide sequence analysis of clones RB-1 and RB-5 was performed bythe dideoxy-terminator method described in Proc. Natl. Acad. Sci.,74:5463-5467 [1977]and yielded the reconstructed complete cDNA sequence.Different deletion templates were generated by the "cyclone" method(Ibid) in single stranded M13 phage clones, which yielded greater than95% of the sequence. The remaining gaps were sequenced by primerextension in both strands. The complete sequence identified in this waycontained 4,523 nucleotides.

An open reading frame was present from the 5' end to base 2688, withnumerous additional in-frame stop codons further downstream. Translationfrom the first methionine codon (base 241) yielded a hypotheticalprotein of 816 amino acids (94,000 daltons in size). The second in-framemethionine was at base 346. Since the nucleotide sequence surroundingthe first ATG is not typical of other known mRNAs (Nucleic Acid Res.,12:857-863 [1984]), the start codon assignment should be regarded astentative. A computer search of the National Biological ResearchFoundation protein sequence database detected no strong homology withany of the more than 4000 published amino acid sequences. However, anumber of nucleic acid-binding proteins and viral proteins showed weaksequence homology, with a yeast DNA-directed RNA polymerase (Cell,42:599-610 [1985]) having the highest homology score.

The predicted protein sequence included ten potential glycosylationsites (CRC Crit. Rev. Biochem., 10:307-366 [1981]) but a candidatetransmembrane domain (at least 20 consecutive hydrophobic residues) wasnot found. The amino acid hydropathy plot showed a mildly hydrophobicregion near the putative amino terminus and a hydrophilic region at thecarboxyl terminus. Two pairs of short amino acid sequences wereidentified that were bracketed by cysteine and histidine residues in themanner of metal-binding domains found in nucleic acid-binding proteins(Science, 232:485-487 [1986]). A region of 54 amino acids from position663 to 716 contains 14 proline residues or about 26% such proline-richregions have also been observed in nuclear oncogene proteins myc and myb(RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor[1985]). While the significance of these observations is not wellestablished, they suggest that the RB gene product may be a nucleicacid-binding protein.

Taking into consideration the previous findings, and the fact that thenucleotide sequence analysis of RB cDNA clones demonstrated a long-openreading frame encoding a hypothetical protein with features suggestiveof a DNA binding function, it was an initial object of this invention toidentify and characterize the RB protein to be used as an antigen forobtaining specific antibody and to determine its predicted DNA binding.

RB protein antigen was prepared by expressing the fusion protein in E.coli. For that purpose, a 20 kb polypeptide fragment of the RB gene wasfused with TRYP E protein and the fusion protein has been expressed inE. coli.

From the hypothetical RB protein sequence data with 816 amino acids andmolecular weight of approximately 98 kD, three pATH plasmids thatexpress TRYP E fusion protein were constructed. The complete cDNA clonewas divided into three portions, namely into fragments 0.7 kb, 0.9 kband 1.8 kb. These three fragments contained the coding sequence of RBcDNA. Plasmid pATH3-0.9 RB was constructed from the fragment 5'0.9 kbinserted into EcoRI-EcoRI site of pATH3. Plasmid pATH3-0.7 RB wasconstructed by inserting middle 0.7 kb fragment of RB-1 clone intoEcoRI-EcoRI site of pATH3, and the plasmid pATH3-1.8 RB was constructedby inserting 3'1.8 kb fragment into BglII-BglII site of pATH3 vector.Orientation was confirmed by detail mapping of the restriction enzymesites.

The recombinant plasmids pATH3-0.9 RB, pATH3-0.7 RB and pATH3-1.8 RBwere then transformed into E. coli mm 294 and grown in M9 minimal mediumwhich was supplemented with tryptophan preferably of concentration ofabout 20 mg/ml. The culture mixture was diluted from 1:10 to 1:150,preferably 1:100, with M9 medium, with casamino acids and ampicillinadded. The procedure for recombinant plasmid construction is describedin J. Virol, 9:132-141 [1984]. The fusion of the fragments into pATHvector frames at the site of restriction enzymes is describes in Proc.Natl. Acad. Sci., 83:4685-4689 [1986].

Only one of the three pATH3 constructs, namely pATH3-0.7 RB expressedthe fusion protein. The obtained fusion protein had a molecular weightof 57 kD. Since the molecular weight of TRYP E is known to be 37 kD, 20kD protein portion of the fusion protein was derived from the RB.

The other two plasmid constructs produced no protein at all, not evenTRYP E itself. Since RB clones contain many hypothetical proteasecleavage sites, the inability to produce protein in E. coli was notsurprising and was probably due to instability of the fusion protein.

Using the above described procedure for fusing pATH3 with RB fragment,large quantities of the fusion protein were prepared and purified bypreparative SDS polyacrylamide gel electrophoresis according toprocedure described in Nature, 227:680-685 [1970]. The fusion proteinwas eluted by overnight extraction and SDS and soluble acrylamide wereremoved by dialysis. The proteins were then concentrated.

Purified fusion protein was used as an antigen in generating specificanti-RB protein anti-body.

The specific rabbit polyclonal antibody against RB protein was preparedby the procedure described generally in Proc. Natl. Acad. Sci.,83:6790-6794 [1986].

Rabbits were repeatedly injected, preferably at 14 day intervals with1-20 μg, preferable 10 μg, of purified fusion protein mixed withcomplete Freund's adjuvant (initial injection) and then given boosterinjections of the same amount of the fusion protein in incompleteFreund's adjuvant (repeated injections). Complete Freund's adjuvantgenerally consists of an emulsion of the antigen, in this case thefusion protein, in saline and a mixture of an emulisifying agent, suchas for example Arlacel A, in mineral oil with killed mycobacteria.Incomplete Freund's adjuvant is the same except that it does not havethe mycobacteria.

The injections were repeated until sufficiently high titer ofanti-fusion protein was detected, approximately for two months, to reactwith both TRYP E and the fusion protein. To enrich for antibodiesrecognizing only RB determinants, two or more affinity columns wereprepared using a methods generally described in Proc. Natl. Acad. Sci.,37:575-578 [1951], and in Immunoadsorbents in Protein Purification,Scand. J. Immunol., Suppl 3 [1976]. At least one column was loaded withTRYP E protein and at least one column was loaded with the fusionprotein. Both columns were appropriately precycled. Antibody was passedfirst through the fusion protein-Sepharose column and eluted withglycine buffer of pH 2.3. The eluate was neutralized and passed throughthe TRYP E column several times to remove antibody specifically directedagainst TRYP E. The purified anti-RB IgG antibody was used forimmunoprecipitation or immunostaining, for localization of RB proteinand will be equally useful for diagnostic identification of RB proteinin human tissue samples.

To identify the RB protein, several human cell lines known to haveeither normal or altered RB gene expression were selected.

LAN-1 neuroblastoma cell line normal human fibroblasts, human hepatomaAlexander cell line and osterosarcoma U2OS cell line were used aspositive controls containing normal RB mRNA. All these cells wereobtained from the American Type Culture Collection (ATCC), Inc.depository. Cell lines with expected shortened or absent RB mRNA, suchas retinoblastomas cell lines Y79 (ATCC), RB355 (gift from RobertPhilips, Toronto, Canada), WERI-1, WERI-24, and WERI-27 (gift from T.Sery Wills' Eye hospital, Philidelphia) were used as negative controls.

All normal human cell lines as described above and all cells from fiveretinoblastomas were labeled with ³⁵ S-methionine and immunoprecipitatedwith preimmune rabbit antibody IgG or rabbit anti-RB IgG.

Cells from all human cell lines were metabolically labeled with ³⁵S-methionine according to the procedure described in J. Virol,38:1064-1076 [1981]. Labeled cell mixtures were immunoprecipitated with1-20 ul, preferably 10 ul, of from 50 ug/ml-200 ug/ml, preferably 100ug/ml of anti-RB antibody IgG using the procedure described in J. Virol,38:1064-1076 [1981].

In all control cell lines a protein doublet with apparent molecularweight of 110-114 kD was detected. In retinoblastoma cell lines, or incells immunoprecipitated with preimmune serum the protein doublet wasnot detected.

The RB proteins immunoprecipitated with rabbit antiRB IgG were analyzedby SDS/polyacrylamide gel electrophoresis and into autoradiography. Theresults are shown in FIG. 1. The RB protein presence is visible atapproximately 110 kD region in lanes 2-5 which illustrate theimmunoprecipitation of the normal positive, i.e., RB protein containingcell lines labeled with ³⁵ S-methionine. Lane 1 is the control line ofneuroblastoma cell immunoprecipitated with the preimmune serum, hencewithout anti-RB protein antibody, rabbit IgG. Lanes 6-10 are obtained byimmunoprecipitation of labeled ³⁵ S-methionine cell lines from fiveretinoblastomas. There is no RB protein present in any of these celllines.

The absence of antigen detectable RB protein in retinoblastoma cellssupports the notion that oncogenicity by mutant RB genes is achievedthrough complete loss of gene product function even in those cell linescontaining shortened RB mRNAs.

The hypothetical protein predicted from the nucleotide sequence wasexpected to have a molecular weight MW about 98 kD. Theimmunoprecipitated protein has a molecular weight MW about 110-114 kD.Complete RB protein amino acid sequence is illustrated in FIG. 2. Thiscomplete sequence is obtained from the newly reconstructed clone whichcontains the most 5' end stretch missing in the original cDNA clone(Science, 1987). The first and second initiation methionines are boxedand alanine and proline clusters are underlined.

The amino acid sequence (FIG. 2) is written in the abbreviation coderecognized in the art. Single-letter abbreviations for the amino acidresidues are: A=Alanine, C=Cysteine, D=Aspartic acid, E=Glutamic acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan andY=Tyrosine.

RB cDNA sequence (Science, 235:1394-1399 [1987]) contained a long openreading frame from nucleotide 1 through 2688, which when translated fromthe first methionine codon yielded a hypothetical protein of 816 aminoacids and molecular weight 98 kD. Recently another isolated RB cDNAclone contained an additional 234 base pairs on the 5' end. The revisedRB cDNA sequence (FIG. 2) still maintained the same open reading frameas in the original clones, and an additional methionine codon was foundat nucleotide 139. When this methionine was used as an initiation codon,the predicted RB protein had 928 amino acids and a molecular weight of110 kD--identical to the apparent molecular weight determined bySDS-PAGE. The additional 5' sequence contains a GC-rich region thattranslates into an unusual cluster of alanine and proline residues (FIG.2).

Discrepancies between actual and apparent molecular weights on SDS-PAGEmay be explained by secondary protein modifications. Several potentialN-linked glycosylation sites are present in the predicted amino acidsequence seen in FIG. 2. However, when LAN-1 cellls were grown in mediumsupplemented with 14galactose or ³ H-glucosamine, labeled RB protein wasnot detected despite prolonged autoradiography. In addition, digestionof ³⁵ S-labeled RB protein by Endoglycosidase H according to methoddescribed in J. Biol. Chem., 250:8569-8579 [1975], did not result in areduction of apparent molecular weight..

When the neuroblastoma cells LAN-1 were metabolically labeled with ³²P-phosphoric acid and immunoprecipitated, the immunoprecipated proteinran as a single band with molecular weight indentical to the ³⁵S-labeled RB protein. The results, illustrated in FIG. 3, show Lanes 2+3showing a ³⁵ S-labeled band at 110-114 kD and Lane 5--³² p-labeled bandat 110-114 kD. Lanes 1 (³⁵ S) and 4 (³² p) are immunoprecipitated withpreimmune rabbit IgG. When the aliquots of RB samples labeled with ³⁵S-methionine were digested overnight with Endoglycosidase H, there wasno detectable reduction of molecular weight 110-114 kD. The abovefindings prove that the retinoblastoma is a phosphoprotein of molecularweight 110-114 kD. The phosphoprotein was therefore named ppRB¹¹⁰.

The RB gene was detected in other vertebrates at the DNA level (Science,235:1394-1399 [1987]) suggesting that the RB gene is present in manyspecies during the evolution and further suggesting an importantphysiological role.

The cells from several vertebrate species, such as QT6 (quail), NIH/3T3(mouse), Rat-2 (Rat) and Cos (monkey) were labeled with ³² P-phosphoricacid as described previously and proteins were immunoprecipitated withantiRB IgG using the same procedure as used for human cells. As shown inFIG. 4, antigenically related proteins were detected in all cells withapparent similar molecular weights of 108 kD in quail, 120 kD in mouse,128 kD in rat and 108-110 kD in monkey, as compared to 110-114 kD inhuman cells.

Antigenically related proteins with varied molecular weights observed indifferent vertebrate species such as quails, mice, rats and monkeyssuggest that the RB protein is conserved through evaluation mostprobably in proportion to evolutionary relatedness. Since both antigensand molecular weights are simultaneously conserved in these vertebratespecies, it is likely that the RB gene product is present andfunctionally similar in other species as well.

The predicted whole amino acid sequence of the ppRB¹¹⁰ protein hasseveral characteristics similar to those appearing in other oncogenes.Therefore, the subcellular localization of the ppRB¹¹⁰ was investigatedby cellular fractionization.

Two methods were employed to find out the distribution of ppRB¹¹⁰between the nuclear, cytoplasmic, or cell membrane fractions.

In agreement with the chemical characterization the ppRB¹¹⁰ sequencesuggesting possible DNA binding, it was determined that 85% of ppRB¹¹⁰is found in the nuclear fraction, with a proportionally small amount(less than 10%) of the ppRB¹¹⁰ located in membrane. There was nodetectable presence of ppRB¹¹⁰ in the cytoplasmic fraction.

To further substantiate that the ppRB¹¹⁰ is localized primarily in thenucleus, the osteosarcoma cell line U20S known to have an advantageouscell morphology for immunohistochemical staining was used. As anexperimental group, the U2OS cells were immunoprecipitated withanti-ppRB¹¹⁰ IgG. As a control group, the U20S cells wereimmunoprecipitated with preimmune IgG. Both groups were then incubatedwith rhodamine conjugated goat anti-rabbit IgG obtained commerciallyfrom Sigma. Immunofluorescence was observed in cells reacted withanti-ppRB¹¹⁰ IgG, namely in the cell nucleus (FIG. 5, Bi). Cells reactedwith preimmune control did not show any fluorescence (FIG. 5, Bii).

The subcellular localization of ppRE¹¹⁰ in the nuclear fraction suggeststhat the RB protein plays an important regulatory function in regulatingother genes and has a DNA - binding activity.

Certain cell lines, particularly those from tumors others thanretinoblastoma, such as neuroblastoma LAN-1 cells were radioactivelylabeled with ³² P-phosphoric acid. Cellular lysates of these labeledcell mixtures were separated by single or double stranded calf thymusDNA cellulose columns according to the method. described in Mol. Cell.Biol., 6:4450-4457 [1986].

The results obtained suggest that the ppRB¹¹⁰ binds only to a limitednumber.of DNA sites that are easily saturated. It has been previouslyshown that other protooncogenes such as c-myc, n-myc, c-myb and c-fosare nuclear phosphoproteins with DNA binding activity (Mol. Cell. Biol.,6:4450-4457 [1986]), Nature, 296:262-266 [1982]. Oncogenic activation ofthese proto-oncogenes occurs by deregulation of gene expression or bystructural modification, and the gene product is essential foroncogenicity.

The ppRB¹¹⁰ absence, and not the presence, appears to be oncogenic dueto the partial or complete inactivation of the RB gene. Therefore, thepresence of the ppRB¹¹⁰ somehow suppresses the oncogenic activity ofother genes and disallows the malignant cell growth. The ppRB¹¹⁰ is thusan important regulatory protein which may prevent and inhibit, by itspresence, and trigger, by its absence, the malignant growth. Thus, theppRBl¹¹⁰ 's importance is in regulating other genes. The absence or lossof ppRB¹¹⁰ mediates oncogenicity.

The utility of the current invention is several fold.

First, the presence or absence of the ppRB¹¹⁰ shall serve as adiagnostic tool in determination of presence or predisposition to theretinoblastoma and other RB gene involved tumors of the human and animalfetus, embryo or newborn babies. Such early diagnosis will allow anearly warning and treatment of retinoblastoma and other tumors with thepossibility of preventing development of the secondary tumorigenesis.

In practice, the use of this invention to diagnose the presence of orpredisposition to retinoblastoma, will be through immunoscreening of thetissue biopsy with specific anti-ppRB¹¹⁰ antibody. The bioptic tissue:will be radioactively labeled with ³⁵ S-methionine, ³² P-phosphoric acidor any other convenient radioisotope and immunoscreened, as describedabove or the proteins extracted from bioptic tissue were blotted onnitrocellulose filter and probed with labeled antibody according tomethods known in the art such as Western blotting.

It is expected, that such readily available diagnostic methods will beused particularly for screening families with a history of hereditaryretinoblastoma. The diagnostic method, however, is also intended to beused for prophylactic prenatal and postnatal screening. Moreover, thediagnostic method will be useful also for prediction of the developmentof secondary cancer, such as for example osteosarcoma, fibrosarcoma,glioblastoma, breast cancer, etc., whether or not connected withretinoblastoma.

The other intended use is for tumorigenesis suppression where the absentppRB¹¹⁰ will be provided through the molecular induction and genetransplanting of the RB cDNA to the individual in need of ppRB¹¹⁰.

Still another use of the current invention is the suppression of thecancerous growth providing intact the RB gene directly to the tumorcells, which cells in turn will produce ppRB¹¹⁰ which will affect theother tumorous cells.

BRIEF DESCRIPTIONN OF FIGURES

FIG. 1 is the chromatogram showing RB proteins in various cell lines.

FIG. 2 is the complete RB cDNA nucleotide sequence and predicted aminoacid sequence of the RB protein.

FIG. 3 is the chromatogram illustrating the immunoprecipitation of theRB gene product.

FIG. 4 is the chromatogram showing a conservation of the RB gene productin different vertebrates.

FIG. 5A is the chromatogram showing the subcellular localization of theRB protein.

FIG. 5B is the immunofluorescence chromatogram showing the RB proteinlocalization within osteosarcoma cell line U20S.

FIG. 6A is the picture of the column chromatography of the RB geneprotein on single stranded DNA cellulose.

FIG. 6B is the picture of the column chromatography of the RB geneprotein on double stranded DNA cellulose.

FIG. 7A is the drawing showing a production of TRYP E-RB fusion protein.

FIG. 7B is the picture of the polyacrylamide gel electrophoresis of theTYRP E-RB fusion protein.

FIG. 8A is the chromatogram showing a immunoprecipitation of RB proteinwith anti-RB IgG of the several human cell lines.

FIG. 8B is the chromatogram showing immunoprecipitation of RB proteinwith anti-RB IgG in retinoblastoma cell lines.

FIG. 9 is the chromatogram illustrating the biochemical fractionation ofthe RB protein.

DETAILED DESCRIPTION OF FIGURES

FIG. 1 is the chromatogram illustrating the identification of RBproteins by immunoprecipitation with rabbit anti-RB IgG in various celllines. Human cells such as neuroblastoma LAN-1 (Lanes 1 and 2),Alexander hepatoma (Lane 3), osteosarcoma U2OS (Lane 4), normalfibroblasts (Lane 5), and five retinoblastomas (Lanes 6 to 10) werelabeled with ³⁵ S-methionine and immunoprecipitated with preimmunerabbit IgG (Lane 1) or rabbit anti-RB IgG (Lanes 2-10). Theimmunoprecipitates were analyzed by 7.5% SDS-polyacrylamide gelelectrophoresis and autoradiographed.

FIG. 2 is the complete RB cDNA nucleotide sequences and predicted aminoacid sequences of the RB protein. The most 5'˜240 nucleotides wereobtained from a cDNA clone from retinoblastoma cell line Y79. Nucleotidesequences from this clone and the original normal RB clones were alignedby sequence overlap. The first and second initiation sites are boxed,and alanine and proline clusters underlined.

FIG. 3 is the chromatogram illustrating the modifications of the RBprotein. LAN-1 cells were labeled with ³⁵ S-methionine (lanes 1-3) or ³²P-phosphoric acid (0.5 mci/ml) (lanes 4 and 5) for three hours. Cellularlysates were immunoprecipitated with preimmune rabbit IgG (lane 1 and 4) or anti-RB IgG (lanes 2, 3 and 5 ) . Aliquots of ³⁵S-methionine-labeled RB proteins were digested with Endoglycosidase H(ICN ImmunoBiologicals) overnight (lane 3). These immunoprecipitateswere then analyzed by 7.5% SDS-polyacrylamide gel as described inFIG. 1. The RB gene product was found to be phosphorylated but notglycosylated.

FIG. 4 is the chromatogram illustrating a conservation of the RB geneproduct in different vertebrate species. Cell lines of humanneuroblastoma, LAN-5, (Lanes 1 and 2), monkey, cos, (lanes 3 and 4),quail fibroblast, QT6, (Lanes 5 and 6), mouse fibroblast, NIH/3T3,(Lanes 7 and 8), and rat fibroblast, rat-2, (Lanes 9 and 10) werelabeled with ³² P-phosphoric acid and immunoprecipitated with preimmuneIgG (odd numbered lanes) or anti-RB IgG (even numbered lanes) andanalyzed as described in FIG. 1. RB proteins of similar butdistinguishable sizes were found among different vertebrate pieces.

FIG. 5A is the chromatogram showing a localization of the RB protein. ³⁵S-methionine labeled LAN-1 cells (Lane 4) were fractionated intomembrane (Lane 1), cytoplasm (Lane 2) and nucleus (Lane 3) and proteinwas immunoprecipitated with anti-RB IgG. The immunoprecipitates werethen analyzed by SDS-PAGE as described for FIG. 1.

FIG. 5B is the chromatogram showing the results of theimmunofluorescence studies of RB protein localization withinosteosarcoma cell line U2OS. Cells reacted with (i) anti-RB IgG and (ii)preimmune rabbit IgG. Most fluorescence was found within the nucleus.

FIG. 6A is the picture of the column chromatography of the RB genephosphoprotein in single stranded DNA cellulose. Protein lysates of thehuman neuroblastoma cells LAN-1 were metabolically labeled with ³²P-phosphoric acid and passed through a single stranded DNA columns andeluted with increasing gradient of NaCl (Lane 3=0.05; Lane 4=0.1; Line5=0.2; Lane 6=0.3; Lane 7=0.5 and Lane 8=1.0 M NaCl). Lane 1 shows thewhole cell lysate immunoprecipitated with anti-RB IgG.

FIG. 6B is the picture of the column chromatography of the RB genephosphoprotein in double stranded DNA cellulose. Protein lysates of thehuman neuroblastoma cells LAN-1 were metabolically labeled with ³²P-phosphoric acid and passed through a double stranded DNA columns andeluted with increasing gradient of NaCl (Line 3=0.05; Line 4=0.1; Line5=0.2; Line 6=0.3; Line 7=0.5 and Line 8=1.0 M NaCl). Line 1 shows thewhole cell lysate immunoprecipitated with anti-RB IgG.

FIG. 7A is the drawing illustrating the production of the TRYP E-RBfusion protein. EcoR1-EcoR1 cDNA RB fragment (0.7 kb) was fused in-frameinto the EcoR1 site of pATH3 vector. Orientation was confirmed bydetailed restriction enzyme mapping. The recombinant plasmid was thentransformed into E. coli mm294.

FIG. 7B is the picture of the polyacrylamide gel electrophoresis of therecombinant TRYP E-RB fusion protein. The recombinant plasmid was thentransformed into E. coli mm294, and grown in M9 minimal mediumsupplemented with 20 mg/ml of tryptophan. The culture was diluted to1:100 in M9 plus casamino acids and ampicillin. At an optical density at600 nm of 0.2, a 1:1000 dilution of a 10 mg/ml stock of indoleacrylicacid in 100% ethanol was added to induce the expression of the TRYP Epromoter. Bacteria cells were pelleted and boiled in Laemmli gel samplebuffer for 15 minutes and analyzed by polyacrylamide gelelectrophoresis. Gel was then stained with Coomassie blue. A 58 kDprotein was found in induction culture (Lane 2) but not in controlculture (Lane 1).

FIG. 8A is the chromatogram showing immunoprecipitation of RBprotein/anti-RB protein IgG from the various human cell lines. ³⁵S-methionine-labeled cells extracts prepared from a human hepatomaAlexander cell line (Lane 1), human osteosarcoma cell line, U2OS (Lane2), normal human fibroblast (Lane 3), human neuroblastoma cell line,LAN-5 (Lane 4), and from neuroblastoma lysates precipitated by preimmunerabbit IgG (Lane 5) were immunoprecipitated with purified rabbit anti-RBIgG. Doublet bands with apparent molecular weight about 110-114 kD wereobserved in Lanes 1-4.

FIG. 8B is the chromatogram showing immunoprecipitation of RBprotein/anti-RB protein IgG from the several retinoblastoma cells.

Cell extracts from five different retinoblastoma cell lines were labeledwith ³² S-methionine and immunoprecipitated with purified rabbit anti-RBIgG. Doublet bands present in cell lines from hepatoma, osteosarcoma,fibroblastoma and neuroblastoma were absent in all five retinoblastomacell lines RB 355 (Lane 1), Y79 (Lane 2), WERI-1 (Lane 3), WERI-24 (Lane4), and WERI-27 (Lane 5), human neuroblastoma cell line LAN-5 (Lane 6)and from neuroblastoma lysates precipitated by preimmune rabbit IgG(Lane 7). The RB protein was identified based on these results.

FIG. 9 is the chromatogram showing a biochemical fractionation todemonstrate the localization of the RB protein. ³⁵ S-methionine labeledwhole cells of LAN 5 (Lane 4) was fractionated into membrane (Lane 1),cytoplasm (Lane 2) and nucleus (Lane 3) and were subsequentlyimmunoprecipitated with rabbit anti-RB IgG. The majority of theRB¹¹⁰⁻¹¹⁴ protein was found in the nucleus with minor portions inmembrane or cytoplasm.

The following examples further illustrate and present a preferredembodiments of the invention disclosed herein. The examples are merelyillustrative of and are not intended to limit the scope of theinvention.

EXAMPLE 1 Preparation of Recombinant Fusion Protein

Recombinant fusion protein has been prepared for use as an antigen forimmunization.

The conserved 5' 0.9 kb, middle 0.7 kb and 3' 1.8 kb regions of RB cDNAwere subcloned into an inducible, high-level TRYP E expression vectors,pATH-3 (University of California, San Diego) using a standard proceduredescribed in Molecular cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

Three RB cDNA subfragments containing the coding sequence, namely 5' 0.9kb (EcoRI-EcoRI), middle 0.7 kb (EcoRI-EcoRI of RB-1) and 3' 1.8 kb(BglII-BglII) were fused in frame with pATH3 vectors, respectively. TheTRYP E-RB gene product of pATH3-0.7 plasmid was expressed in E. coliusing a method described in J. Virol, 49:132-141 [1984]. pATH3-0.7 RBwas constructed as illustrated in drawing 7A. cDNA RB fragment wasinserted in-frame into the EcoRI endonuclease site of pATH-3 plasmid.Orientation was confirmed by detail restriction enzyme mapping. Therecombinant plasmid was transformed into E. coli mm294 and grown in M9minimal medium supplemented with 20 mg/ml of tryptophan. The culture wasdiluted to 1:100 in M9 plus casamino acids and ampicillin. At an opticaldensity at 600 nm of 0.2, a 1:1000 dilution of a 10 mg/ml stock ofindoleacrylic acid in 100% ethanol was added to induce the expression ofthe TRYP E promoter.

Bacteria cells were pelleted and boiled in Laemmli gel sample buffer for15 minutes and analyzed by polyacrylamide gel electrophoresis. Gel wasthen stained with Coomassie blue. Large quantities of fusion proteinwere prepared and purified through preparative polyacrylamide gelelectrophoresis and eluted by overnight extraction. SDS and solubleacrylamide were removed by dialysis. The proteins were then concentratedand mixed with adjuvant for immunization of rabbits. About 6 mg proteinwas recovered which was subsequently used for immunization of rabbits.

The expression in E. coli produced, after induction, a 57 kD fusionprotein comprising 20% of total E. coli protein. pATH-0.7 expressed afusion protein with molecular weight of 57 kD, of which 37 kD was TRYP Eand 20 kD was RB-derived. The other two plasmids produced no protein atall.

Production of the TRYP E-RB fusion protein can be seen in FIG. 7A andthe results of the SDS/PACE electrophoresis in 7B. A 58 kD protein wasfound in induction culture (Lane 2) but not control culture (Lane 2).

EXAMPLE 2 Anti ppRB¹¹⁰ --Specific Antibody

Two rabbits were immunized with the TRYP E ppRB¹¹⁰ fusion proteinobtained in Example 1 following a standard protocol.

New Zealand Red rabbits were initially injected with 10 μg of purifiedTRYP E ppRB¹¹⁰ fusion protein mixed with complete Freund's adjuvant andthen given booster injections of 10 μg of TRYP E ppRB¹¹⁰ fusion proteinin incomplete Freund's adjuvant. The booster injections were repeatedfor several months until high titers of antibody were detected byimmunoprecipitation analysis according to Proc. Natl. Acad. Sci.,76:4350-4354 [1979].

Two months later, both rabbits produced high titers of antibodies thatreacted with both TRYP E and the fusion protein. The rabbits were bledand the blood was collected into plastic containers and clotted. Theserum was obtained by centrifugation at 1000 g for 10 minutes. Rabbitanti-ppRB¹¹⁰ immunoglobulin (IgG) was purified by passing the antiserathrough the two affinity columns. To enrich for antibodies recognizingonly RB determinants, two affinity columns were prepared, one with TRYPE protein and the other with the fusion protein. The antisera was passedthrough the fusion protein-Sepharose column and eluted with 0.1M glycineHCl buffer (pH 2.3). The eluate was passed through the TRYP E columnseveral times to remove antibody directed against TRYP E using the samebuffer. The elution was repeated several times. Antibody preparedthrough the above steps was serially diluted and the dilution sufficientfor immunoprecipitation of RB protein in 1.5×10⁶ cells were determined.This purified anti-RB antibody was used in all subsequent experimentsfor immunoprecipitation or immunostaining and to immunoprecipitate RBprotein in several cell lines which were previously demonstrated tocontain intact RB mRNA according to procedure of Example 3.

EXAMPLE 3 Immunoprecipitation Identification of RB Protein with Anti-RBAntibody

A standard protocol was followed as described in J. Virol., 38:1064-1076[1981].

LAN-1 neuroblastoma cell line, normal human fibroblasts, human hepatomaAlexander cell line and osterosarcoma U2OS cell line which containnormal RB mRNA were used as positive controls. All these cells wereobtained from the American Type Culture Collection, Inc. depository.Cell lines with expected shortened or absent RB mRNA, such asretinoblastomas cell lines Y79, RB355, WERI-1, WERI-24, and WERI-27 wereused as negative controls. These cell lines were obtained as describedabove.

To label cellular proteins with ³⁵ S-methionine, about 1.5×10⁶ cells in60-mm petri dishes were starved by incubation at 37° C. for 30 minutesin methionine-free medium and then incubated in 3 ml of methionine-freemedium supplemented with ³⁵ S-methionine (150 uCi/ml) for three hours.All subsequent operations were at 4° C. Cell extracts were prepared inlysis buffer containing 25 mM Tris-hydrochloride (pH 7.4), 50 mM NaCl,0.2% Nonidet P-40, 0.5% deoxycholate and 200 units/ml of Aprotinininactivator. 0.02% SDS was also added at the beginning of lysis. Thelysates were clarified by centrifugation at 4° C. at 20,000×g for 15minutes.

Immunoprecipitation was carried out with 5 ul of preimmune rabbitantisera, followed by absorption to formalin-fixed Staphylococus aureusobtained from The Enzyme Center, Inc. To the supernatant of eachexperimental sample was added 10 ul of 100 ug/ml of anti-ppRB¹¹⁰ IgG andto the supernatant of each control sample 10 ul of the preimmune serawas added for control. Protein A sepharose beads (Sigma) were thenadded. Immunoprecipitates were subsequently washed with 1) lysis buffer,2) 1M NaCl in lysis buffer, 3) 0.15M NaCl in lysis buffer, and 4) lysisbuffer to remove nonspecifically bound proteins. The immunoprecipitatedproteins were analyzed by 7.5% SDS-polyacrylamide gel electrophoresisand autoradiographed. Gels of ³⁵ S-labeled proteins were fluorographedat -70° C. after impregnation with acetic acid-based2.5-diphenyloxazole.

The results are illustrated in FIG. 1. The protein with molecular weight110-114 kD was found to be immunoprecipitated with anti-ppRB¹¹⁰ IgG.

EXAMPLE 4 Characterization Of The RB Gene Product

To further characterize the ppRB¹¹⁰ protein, the cells were labeled with³² P-phosphoric acid or with ¹⁴ C or ³ H-glucosamine and subsequentlydigested with Endoglycosidase H.

To test for protein phosphorylation, LAN-1 cells were metabolicallylabeled with ³² P-phosphoric acid. To label LAN-1 cells with ³² p,around 1.5×10⁶ cells in 60-mm petri dishes were starved by incubation at37° C. for 80 minutes in phosphate-free medium and then incubated for 1to 2 hours in 2 ml of phosphate-free medium supplemented with ³² PO₄ ³-(lm ci/ml) medium. Cell extracts were prepared in lysis buffercontaining 25 mM Trishydrochloride of pH 7.4, 50 mM NaCl, 0.2% NonidetP-40, 0.5% deoxycholate and 200 units/ml of kallikrein inactivatorobtained from Calbiochem. The lysate was clarified at 4° C. at 20,000×gfor 20 min.

Immunoprecipitation was carried out with anti-ppRB¹¹⁰ IgG according tostandard procedure. Immunoprecipitate was absorbed to formalin-fixedStaphylococcus aureus obtained from The Enzyme Center and subsequentlywashed with 1) lysis buffer; 2) 1M NaCl, 10 mM Tris-hydrochloride(pH7.4) and 0.1% Nonidet P-40; 3) 0.15 mM NaCl, 10 mM Trishydrochloride(pH 7.4), 0.1% Nonidet P-40; and 4) lysis buffer.

The immunoprecipitated proteins were prepared for electrophoresisfollowing the procedure described in J. Virol, 36:617-621 [1980].

Immunoprecipitated protein ran as a single band with molecular weightidentical to that of ³⁵ S-labeled ppRB¹¹⁰ protein indicating that the RBprotein is a phosphoprotein.

EXAMPLE 5 Subcellular Localization Of The pDRB¹¹⁰

Human neuroblastoma cells LAN-1 were labeled with ³⁵ S-methionine asdescribed in Example 3, and fractionated into membrane, cytoplasm andnucleus. The labeled protein was subsequently immunoprecipitated withanti-RB IgG.

The cell fractionation protocol is essentially adapted from thatdescribed in J. Cell. Biol., 97:1601-1611 [1983].

Two to five 100-mm plates containing a total of 2.0×10⁷ to 7.5×10⁷ LAN-1cells were metabolically labeled with ³⁵ S-methionine for 2-3 hoursprior to use. All subsequent procedures were performed at 4° C. Cellswere rinsed twice with phosphate-buffered saline (PBS), scraped intoPBS, and pelleted for 5 minutes at 375×g in a table top centrifuge. Halfthe cells were resuspended in lysis buffer for the whole cell lysate.The remaining cells were rinsed once in hypotonic RSB buffer (10 mMHEPES pH 6.2, 10 mM NaCl, 1.5 mM MgCl₂, 200 units/ml Aprotinin) and thenresuspended in RSB. The cell suspension was homogenized by 20 strokes ina tight fitting Dounce homogenizer, and volume was adjusted to exactly 3ml with RSB buffer. The homogenate was centrifuged at 1,500 rpm in aSorvall HB4 rotor at 375×g for 10 minutes, and the pellet wasresuspended in lysis buffer to generate the nuclear fraction. Thesupernatant was centrifuged in thick-walled polyallomer tubes in aBeckman SW50.1 rotor at 35,000 rpm (150,000×g) for 90 minutes. Thepellet was resuspended in lysis buffer to generate the membranefraction, while the supernatant was adjusted to 1 X lysis bufferconcentration to produce the cytoplasmic fraction. All four fractionswere then assayed for RB protein content as illustrated in Example 3.

The immunoprecipitates were analyzed by 7.5% SDS-polyacrylamide gelelectrophoresis and autoradiographed.

The results are summarized and illustrated in FIG. 5A.

As an alternative, the following fractionation method was used. Allprocedures were performed at 0° to 4° C. Two to five 100-mm platescontaining a total of 2.0×10⁷ to 7.5×10⁷ LAN-1 cells were rinsed twicewith phosphate-buffered saline (PBS), scraped into PBS, and pelleted for1 min. at 1,000×g in a clinical centrifuge. After the pellet was rinsedonce with hypotonic TKM buffer (20 mm Tris pH7.1, 5 mM KCl, 1 mM MgCl₂,1% Aprotinin), the cells were dispersed and swollen in TKM for 15 min.The cell suspension was homogenized by 20 strokes in a tight fittingDounce homogenizer. The volume was adjusted to exactly 3 ml with TKMbuffer, and samples were removed for analysis by immunoprecipitation.

Nuclear pellet was generated by low-speed centrifugation, and thesupernatant from this initial centrifugation was subjected to high-speedcentrifugation to obtain a precipitate and a soluble fraction. To obtaina nuclear pellet, the homogenate was centrifuged at 1,500 rpm in aSorvall HB4 rotor (375×g) for 10 min. at 0° C., and the crude nuclearpellet was suspended in 1 ml of TKM. This pellet was then homogenizedfive times in the Dounce homogenizer and aspirated three times through a1 ml syringe fitted with a 25-gauge needle. The suspension was thenpelleted as described above, suspended in TKM buffer and aspirated againfive times through the same syringe. After a final centrifugation, thenuclear pellet was suspended in TKM buffer and analyzed for RB proteincontent and for subcellular markers. The original postnuclearsupernatant (PNS) and the supernatants from the nuclear pellet washeswere pooled and centrifuged in thick-walled polyallomer tubes in aBecknan SW50.1 rotor at 38,000 rpm (150,000×g) for 90 minutes at 0° C.to generate particulate (P₁₅₀) and soluble (S₁₅₀) fractions. Thefractions were then adjusted to equal volumes with TKM buffer andassayed directly for RB protein and subcellular markers.

Plasma membrane content was determined by measuring 5' nucleotidase. Thesamples were taken up in TKM buffer and incubated in an assay mixturecontaining 10 mM MgCl₂, 0.1 mM AMP, 100 mM glycine (pH 9.0), and 2 uCiof ³ H-adenosine in the supernatant determined by liquid scintillationcounting (Ibid). The soluble protein in each fraction was determinedassaying for lactate dehydrogenase activity according to Proc. Natl.Acad. Sci., 48:2123-2130 [1962], and the endoplasmic reticulum contentwas measured with an assay for NADH diaphorase according to proceduredescribed in Biochem. Biophys. Acta., 233:334-347 [1971].

The results are illustrated in FIG. 9. Using methods of biochemicalfractionation and immunofluorescence (Example 6), the RB protein wasdetermined to be localized primarily in the nucleus.

A majority (about 85%) of ³⁵ S-labeled protein was located in thenuclear fraction while a minor portion (less than 10%) was associatedwith membrane. There was no detectable RB protein within the cytoplasmicfraction, or secreted into the medium.

EXAMPLE 6 Subcellular Localization of ppRB¹¹⁰ Measured .ByImmunofluorescence

Human osteosarcoma cell line U20S, obtained from American Type CultureCollection, Inc., were used for immunofluorescent staining.

About 10⁴ U2OS cells were seeded onto 12-mm glass cover slips and used18 hours later for immunofluorescent staining. The cells were washedonce with PBS buffer and fixed with cold acetone for 10 minutes at roomtemperature. Fixed and permeabilized cells were hydrated in PBS for 1 to2 minutes. Each cover slip was incubated with 200 ul of rabbit anti-RBIgG (1:20 dilution) or preimmune serum in a moist chamber for 45 minutesat room temperature. After three washes in PBS, the cover slips wereincubated with 200 ul of rhodamine-conjugated goat anti-rabbit IgG (25ug/ml) obtained from Sigma for 45 minutes at room temperature. The coverslips were again washed extensively in PBS and viewed with a Zeissphotomicroscope III.

Alternatively, immunofluorescent staining of LAN-1 neuroblastoma celllines was carried out as follows. 10⁴ LAN-1 cells were seeded onto 12-mmglass cover slips and used 18 hours later for immunofluorescentstaining. The cells were washed once with PHEM buffer consisting of 0.06M Pipes, 0,025M HEPES, 0.01M EGTA, 0.002M MgCl₂, pH 6.9 and fixed with2% paraformaldehyde in PHEM buffer for 20 minutes at room temperature.Fixed and permeabilized cells were washed once in PHEM buffer and threetimes in PBS. Each cover slip was incubated with 12 ul of a 1:80dilution of a rabbit anti-RB IgG, or preimmune serum in a moist chamberfor 45 minutes at room temperature. After three washes in PBS, the coverslips were incubated with 12 ul (25 ug/ml) of fluorescein isothiocyanateconjugated goat and anti-rabbit immunoglobulin G obtained from SigmaChemical Co., for 45 minutes at room temperature. The cover slips wereagain washed extensively in PBS and incubated at room temperature for 45minutes with rhodamine-conjugated phalloidin (20 ug/ml). The stainedpreparation was mounted in PBS-glycerol (1:9) containing theantibleaching agent p-phenylenediamine and viewed with a Zeissphotomicroscope III.

The results are illustrated in FIG. 5B and are similar to those obtainedby biochemical fractionation described in Example 5. The fluorescencewas present mainly within the nucleus and the preimmune control wasnegative.

EXAMPLE 7 DNA Binding Activity Assay

Two DNA binding assays, DNA-Sepharose column chromotography and filterbinding, were used.

These two methods have been previously used in studies of myc (Nature,296:262-266 [1982]), N-myc (Embo J., 4:2627-2633 [1985]), and mybproteins (Cell, 40:983-990 [1985]).

Sepharose Column Chromatography

To test DNA binding activity of the RB protein, double-stranded andsingle-stranded calf thymus DNA coupled onto Sepharose 2B obtained fromPharmacia was employed.

Protein lysates of the human neuroblastoma LAN-1 cells weremetabolically labeled with ³² P-phosphoric acid and separated onsingle-stranded (A) and double-stranded (B) DNA cellulose columns namelywith 0.05, 0.1, 0.2, 0.3, 0.5 and 1.0M NaCl. Column chromatographyseparation of the RB gene protein product on single stranded and doublestranded DNA cellulose is illustrated in FIG. 6. LAN-1 cells labeledwith ³² P-phosphoric acid for three hours were lysed in lysis buffer andclarified as described previously. The supernatant was diluted 10-foldwith loading buffer (1 mM DTT, 0.5% NP40, 10 mM potassium phosphate, 10%glycerol pH 6.2, Aprotinin 200 units/ml). The diluted extract was thenapplied to calf-thymus DNA-cellulose columns (Pharmacia) Mol. CellBiol., 6:4450-4457 [1986], equilibrated with loading buffer containing50 mM NaCl. After allowing binding to occur for 40 minutes, the columnwas washed with 5 ml of loading buffer and then eluted with buffercontaining 1 mM DTT, 10 mM Tris-HCl, pH 8.0 with increasing NaClconcentration from 0.05 to 1.0M. The eluates were thenimmunoprecipitated with rabbit anti-RB IgG. The whole cell lysates andflow-through immunoprecipitated with anti-RB IgG, respectively, servedas the controls. Fractions were then analyzed by immunoprecipitation asdescribed in Example 3.

The results are illustrated in FIG. 6.

EXAMPLE 8 Diagnostic Determination of the ppRB¹¹⁰ in the Tissue

Tumor cells disassociated from biopsy tissue from the subject waslabeled with ³⁵ S methionine or ³² P-phosphoric acid animmunoprecipitated with anti-ppRB¹¹⁰ IgG according to the procedure ofExample 3. Alternatively, protein lysates extracted from biopsy tissuecan be directly diagnosed using the Western blotting analysis probedwith either radioactive labeled or non-radioactive labeled anti-RBspecific antibody. The presence or absence of immunoprecipitatedproteins serves as a diagnostic tool in determination of retinoblastomaor other diseases controlled by the retinoblastoma gene.

What is claimed is:
 1. A purified polyclonal antibody which specificallybinds retinoblastoma phosphoprotein ppRB¹¹⁰.
 2. The antibody of claim 1which is radiolabeled.
 3. The antibody of claim 2 having the ability toimmunoprocipitate the retinoblastoma phosphoprotein ppRB¹¹⁰.
 4. Theantibody of claim 3 which can form an immunocomplex with ¹²⁵ I labeledprotein-A.